Mechanism of network pharmacology of Erzhi Pill and Erxian Decoction in treating climacteric syndrome with “treating the same disease with different methods”: A review

Network pharmacology and molecular docking methods were used in the present study to clarify the molecular mechanism of two traditional Chinese medicine prescriptions of climacteric syndrome. Based on oral availability and drug similarity, the main active components of Erzhi Pill and Erxian Decoction were screened through the platform of traditional Chinese medicine system pharmacology. The target database of climacteric syndrome was established by using GENECARD, OMIM, PharmGKB, Targets and Drugbank. The “component – target” network diagram was constructed using Cytoscape software (version 3.8.2). Topology analysis, module analysis, and GO and KEGG enrichment analyses were used to explore the core target and action pathway of Erzhi Pill-Erxian Decoction for treating climacteric syndrome of same disease with different treatments. There were 16 active components and 103 corresponding targets found in Erzhi Pill; 69 active components and 121 corresponding targets were found in Erxian Decoction; and 100 potential targets were found in Erzhi Pill and Erxian Decoction. Through network analysis, topology and module analysis, TP53, AKT1, Jun, ESR1, IL1B, CASP3, MMP9, PTGS2, HIF1A, MYC and EGFR could be considered as potential targets of the 2 prescriptions for alleviating climacteric syndrome. The effects of Erzhi pill and Erxian Decoction on climacteric syndrome are mainly in the pathway of lipid and atherosclerosis, AGE-RAGE signaling pathway and PI3K-Akt signaling pathway in diabetic complications. The active components in Erzhi Pill - Erxian Decoction, such as quercetin, show considerable potential as a candidate drug for the treatment of climacteric syndrome.


Introduction
Menopausal syndrome refers to a group of syndromes before and after menopause (45-55 years old), due to the fluctuation or decrease of sex hormones caused by the decline of ovarian function, resulting in vasodilatation and autonomic dysfunction and psychoneurological anomalies, with menstrual dysfunctions, hot flashes, sweating, insomnia, emotional anomalies, and bone, joint, and muscular pains as the main manifestations, which is also known as the "perimenopausal syndrome."It is also known as "perimenopausal syndrome," and in Chinese medicine, it is called "all the symptoms before and after menopause" and "all the symptoms before and after menstrual break."Its incidence and severity are affected by various factors such as geography, race and individual differences. [1]Menopause is a physiological event in a woman's life and natural part of the aging process.The transition from full ovarian function in the premenopausal period to a complete lack of ovarian estrogen synthesis in the postmenopausal period is a natural process but can greatly influence daily life in women and lead them to seek medical help.For some women, these symptoms may have a significant negative impact on quality of life. [2]Data statistics: in 2010, there were about 160 million climacteric women in China, and more than 120 million women were troubled by climacteric syndrome (CS) every year, which seriously affected the quality of life and work.Improving the quality of life of menopausal women has been listed as one of the 3 major health issues in The study was funded by Zhejiang Province Medical and Health Technology Plan Project (no.2021KY609), Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (no.2020ZB055), and Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (no.2021ZB069).

The authors have no conflicts of interest to disclose.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.a Department of Gynaecology, Tongde Hospital of Zhejiang Province, Hangzhou, China.the 21st century. [3]At present, the treatment of CS by western medicine is still mainly hormone therapy, supplemented by symptomatic treatment. [4,5]However, there are some adverse effects and risks associated with hormone therapy, such as increasing the patient's risk of endometrial cancer, breast cancer, abnormal uterine bleeding, and cardiovascular diseases. [6]here are widespread fears and misunderstandings about hormone preparations in patients, which reduce the compliance of hormone therapy.
There are notable methods of traditional Chinese medicine (TCM) in the treatment of CS.TCM can achieve a good longterm curative effect after dialectical treatment, with minimal side effects.Such treatment has been accepted and recognized by people, and has distinct advantages over modern medicine for the treatment of CS.
Erzhi Pill consists of Ligustrum lucidum and Eclipta, modern studies have found that Erzhi Pill has a wide range of pharmacological activities in perimenopausal syndrome, hepatoprotection, osteoporosis, immune system, anti-aging, and neurological disorders. [7]In gynecological diseases, Erzhi Pill can relieve a series of autonomic nervous system dysfunction syndromes caused by fluctuation or decrease of sex hormones in ovarian function decline, including paroxysmal baking heat and sweating, irritability, palpitation and insomnia, hot flashes, red face, and emotional restlessness, etc. [8] Erxian Decoction is a prescription created by Professor Zhang Bo-Na for treating diseases caused by functional decline of ovary and estrogen decrease. [9]This formula is made up of Xianmao, Epimedium, Bacopa monnieri, Angelica sinensis, Zhimu, and Phellodendron Bark.The primary effects are inhibiting ovarian apoptosis, and improving the estrogen level in the body.Through modern pharmacological research, Erxian Decoction has been found to regulate reproductive endocrine from the levels of gonadotropin, hypothalamic reproductive regulatory hormone, HPOA and ovarian granulosa cells.Further, Erxian Decoction can change the level of reproductive hormone, regulate the balance between the hypothalamic -pituitary -ovarian -uterine female sexual axis in the body, promote female autonomic nerve function, increase immune improvement, and improve a series of CS caused by increased gonadotropin level and decreased estrogen level in women. [10]The combination of both sides can improve the symptoms of patients with CS, enhance their quality of life, and regulate their immune function.For CS, taking into account the use of hormones in western medicine to replace clinical side effects, the balance of yin and yang can be restored through TCM and treating both the manifestation and root cause of disease, thereby improving certain ovarian functions and the level of estrogen.
In TCM, the concept of "treating the same disease with different methods" was proposed thousands of years ago, that is, the same disease can be treated according to different people, times and places, or due to the development of the disease, different disease types and different changes of pathogenesis.Essentially, different treatment methods should be adopted according to different situations. [11]Such concept has gradually become the essence of TCM treatment, and the application characteristics thereof reflect the characteristics of TCM diagnosis and treatment of diseases that emphasize the combination of syndrome differentiation, disease diagnosis and treatment. [12]In the present study, the network pharmacology method was used to construct the network target and pathway combination of Erzhi pill-Erxian Decoction, the key protein for alleviating CS was found, and the action mechanism of "treating the same disease differently" for CS was revealed.The present study provides a basis for explaining the scientific connotation and pharmacological action mechanism of TCM (see Fig. 1).

Screening of CS targets
With "perimenopausal syndrome" or "menopause" as the keyword, data were searched in GenecCards Database (http:// www.genecards.org/), [13]Human Mendelian Genetic Database (OMIM) (http://omim.org/,Update in 2022-07-03), [14] Pharmacogenomics Knowledge Database (PharmGKB) (http:// www.pharmgkb.org/), [15]Drugbank Database (http://www.drugbank.ca/) [16]and Therapeutic Target Database (Therapeutic Targets) ( http://bidd.nus.edu.sg/group/ttd/ttd.asp,TTD Vision 4.3.02). [17]To obtain the data set of disease target genes, the repeated targets were removed after merging.The common targets of CS gene and Erzhi Pill-Erxian Decoction were compared and analyzed with R package, and the intersection targets of disease and drugs were displayed through a Venn diagram.The intersection targets were used as a bridge to construct the regulation network of TCM.This study did not involve human or animal subjects, and thus, no ethical approval was required.The study protocol adhered to the guidelines established by the journal.

Screening of active components and targets of Erzhi Pill and Erxian Decoction
Through TCMSP (https://www.tcmsp-e.com/), [18]the active components and targets with 8 kinds of TCM were searched under the screening conditions of OB (oral bioavailability, OB) ≥ 30% and DL (drug likeness, DL) ≥ 0.18 in Erzhi pill-Erxian Decoction.Subsequently, the species was limited to humans through the protein database (UniProt), and then the genes corresponding to each target were corrected to obtain the standardized name abbreviation (Symbol).

Construction of "active ingredient-target" TCM compound regulation network of Erzhi Pill-Erxian Decoction
Drug active ingredient information, intersection target genes and CS target genes were introduced into Cytoscape 3.8 2 software, [19] the regulation network of "active ingredient-target" was constructed, and the correlation between the Erzhi Pill and Erxian Decoction active ingredients and the disease genes of CS.

Construction of protein-protein interaction network (PPI) of common target of CS gene and Erzhi Pill-Erxian Decoction
The intersection targets obtained in 1.3 were imported into the String Database, [20] the common targets of Erzhi Pill -Erxian Decoction and CS were imported into the multiple proteins function, and the organization was selected."Homo sapiens" was selected to construct the common target PPI of Erzhi Pill -Erxian Decoction and CS, respectively.For the parameter settings, the minimum interaction score was 0.4 by default.Disrupted nodes were hidden in the network.The obtained information was then imported into Cytoscape 3.8.2software in the form of tsv to construct Erzhi Pill, Erxian Decoction and CS, and the common or unique active ingredients of both prescriptions for the climacteric syndrome target points network.Topological and modular analysis could be used to obtain the relationship between Erzhi Pill -Erxian Decoction targets and CS.

Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis
The gene abbreviation (symbol) was transformed into gene ID, and then the GO (Gene ontology) enrichment and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment of "Drug-Disease" gene were analyzed by R software.Subsequently, the relevant action pathway map was drawn.

Verification of molecular docking between active ingredients and target genes
The 2D structure of Erzhi Pill-Erxian Decoction for CS related target active components was obtained in the organic small molecule bioactivity database (PubChem, https://pubchem.ncbi.nlm.nih.gov/), [21]and the 3D structure of the target was obtained in the protein structure database (PDB).PyMOL software was used to complete the treatment of protein receptor structure dehydrator and invalid small molecule ligand.Finally, Vina was used for molecular docking.The binding relationship and binding position between active molecules and receptor protein in Erzhi Pill-Erxian Decoction were obtained.

Screening gene of CS
In the databases of GeneCards, OMIM, PharmGKB, TTD, Drugbank and others were searched with "perimenopausal syndrome" or "menopause" as the keywords.The genes retrieved were filtered with relevance score > 1.0 in the GeneCards database.After searching the retrieved genes in 5 databases, a total of 1383 gene targets of CS was obtained, and then the Venn diagram was drawn (see Fig. 2).

Screening of effective active ingredients of Erzhi Pill -Erxian Decoction
In  1 and 2.

Prediction of "drug target -disease gene" intersection target
Via R x64 4.0 4 Venn analysis was conducted on the gene targets corresponding to the effective active components of Erzhi Pill-Erxian Decoction and the gene targets of CS diseases.Finally, 103 and 121 direct or indirect effective target gene intersections were obtained, respectively.At the same time, through analysis and calculation, the target gene mapping rates of Erzhi Pill and Erxian Decoction for alleviating CS were 55.98% and 57.62%, respectively, indicating that both were specific.The common and unique targets of Erzhi Pill-Erxian Decoction were then sorted, the Venn diagram of common targets of Erzhi Pill-Erxian Decoction and CS was drawn (see Fig. 3), and 100 intersections were obtained.Such findings indicate that there were 100 targets related to CS, that is, the potential targets of both prescriptions for alleviating CS.The specific information of gene targets was then sorted.

Construction, topology and module analysis of "active ingredient target gene" regulatory network
The network diagram of "Erzhi Pill-Erxian Decoction drugs CS targets" was constructed by using Cytoscape software (see Fig. 4).An observation can be made that the network was composed of 124 potential target genes and 81 active components, including 12 specific active components of Erzhi Pill, with 133 targets and 244 edges, 65 specific active components of Erxian Decoction, with 121 targets and 557 edges, and 4 active components of both compounds, with 94 targets and 156 edges.Each potential target gene interacted with multiple active components, which reflected that Erzhi Pill-Erxian Decoction intervened in the treatment of CS through multiple channels, multiple components and multiple targets.
The topological analysis of the top 10 degrees was obtained according to the degree size.The topological analysis of potential targets of Erzhi Pill is shown in Table 3, the topological analysis of potential targets of Erxian Decoction is shown in Table 4, and the topological analysis of potential targets for the same disease and different treatment are shown in Table 5.
According to the topological analysis in the network diagram and table, the potential targets of Erzhi Pill with the top 10 degrees to alleviate CS were AKT1, TP53, JUN, IL1B, ESR1, CASP3, PTGS2, EGFR, MMP9, and MYC (see Table 3); the potential targets of Erxian Decoction for alleviating CS were AKT1, TP53, ESR1, Jun, IL1B, EGFR, CASP3, PTGS2, HIF1A, and MMP9 (see Table 4); and the potential targets of both for alleviating CS were AKT1, TP53, JUN, ESR1, IL1B, CASP3, MMP9, PTGS2, EGFR, and HIF1A (see Table 5).Based on the aforementioned information, the 11 gene targets of TP53, AKT1, JUN, ESR1, IL1B, CASP3, MMP9, PTGS2, HIF1A, MYC, and EGFR were potential targets for both prescriptions to jointly alleviate CS.An observation can be made from Figure 4 that the common active component of Erzhi pill and Erxian Decoction was MOL000098: Quercetin.There was a significant difference due to the high degree value in the network diagram, suggesting that Quercetin could be used to alleviate CS.

Construction of PPI network of intersection target of Erzhi Pill -Erxian Decoction and CS
Using the String11.0online tool, the common targets of the disease for both drugs was imported, in addition to the specific targets of CS for both drugs.There were 99 nodes and 1364   edges in the interaction network of the common targets of both drugs (see Fig. 5A), there were 3 nodes and 2 edges in the CS target interaction network of the specific targets of Erzhi pill (see Fig. 5B), and there were 18 nodes and 24 edges in the CS target interaction network of the specific targets of Erxian Decoction (see Fig. 5C).
The specific targets of Erzhi Pill and Erxian Decoction analyzed by String were introduced into Cytoscape, respectively.Through degree value analysis, the specific targets of Erzhi Pill were found to have higher moderate values for alleviating CS: CYP19A1, and the specific targets of Erxian Decoction were found to have higher moderate values for alleviating CS: SLC6A3, MAOA, DRD2, and NR3C1.
Cytoscape-ClusterViz was used for module analysis, and the MCODE algorithm was selected for cluster analysis of the common targets of both drugs and diseases. [22]The default parameters were set with NodeScore Threshold = 0.2, and K-Core Threshold = 4. K-Core is a parameter that determines the size of the identification module, that is, the edge corresponding to the obtained module should be greater than 4. Two modules were obtained, and the numbers of genes were 40 and 15, respectively, in each module (see Table 6).R was used to analyze the genes corresponding to the module (see Table 7).

Potential target GO enrichment annotation and KEGG pathway enrichment analyses
For Erzhi Pill and Erxian Decoction, the common targets of both prescriptions would become the potential antidepressant targets of "treating the same disease with different methods" for CS.The results of GO and KEGG analysis show that in terms of enrichment results, there were 1933 of biological process, 53 of cell composition, 155 of molecular function and 151 of KEGG pathways.The results indicate that the biological processes of both prescriptions were mainly reflected in the regulation of lipopolysaccharide, molecular of bacterial origin and oxidative stress in alleviating CS.The cell composition was mainly reflected on the cell membrane.The focus of molecular functions was on nuclear receptor activity, transcription factor activity, direct ligand regulated sequence specific DNA binding and steroid hormone receptor activity (see Fig. 6A).The pathway was concentrated in the lipid and atherosclerosis, the AGE-RAGE signaling pathway in diabetic complications and PI3K-Akt signaling pathway (see Fig. 6B and C).In previous studies, [23,24] the PI3K-Akt signaling pathway was found to be related to the onset of CS.The results suggest that CS might also be related to atherosclerosis and dysregulation of neurotransmitter transmission.Erxian Decoction could alleviate CS, mainly focusing on neuroactive ligand receptor interaction and dopaminergic synapse, cGMP-PKG signaling pathway and calcium signaling pathway.The specific targets of Erzhi Pill for alleviating CS are apolipoprotein B (APOB) and FASLG, which also indicates that APOB and FASLG may be different between normal and CS patients.APOB is often used to predict the severity of coronary heart disease clinically, [25] and a recent study demonstrated that blood gene expression of FASLG, a Fas receptor-ligand triggering non-apoptotic inflammatory activities, is overexpressed in males and females after the age of 50, [26] additional studies are encouraged to test the presence of these biomarkers in CS modality.

Molecular docking simulation of active ingredients and target genes
The core target genes in the PPI core network were selected according to the degree value and the relevant literature on gene research of CS.The common active components and 4 molecules of Erzhi Pill and Erxian Decoction were selected through the "active component target gene" network of Erzhi Pill and Erxian Decoction.The docking results of key targets and key components were mostly lower than −5.0 kcal/mol, [27] suggesting that the key components and the key targets formed a stable structure.The docking results are shown in Table 8.PyMOL software was used to visualize the docking results of the active components with the strongest binding activity of the target gene.The results are shown in Figure 7.

Discussion
With the recent acceleration in the aging of the global population, the prevention and treatment of CS has become increasingly severe.The has also been a surge of interest in the research on Erzhi pill and Erxian Decoction as representative prescriptions for relieving CS.Regarding Erzhi Pill and Erxian Decoction as treatments to alleviate menopause separately, several researchers have predicted that their elaboration on the mechanism of CS often focuses on the perspective of "point" or "line" or "body."Meanwhile, "point -line -surface -body" is rarely integrated  to clarify the mechanism of treating CS.In TCM, most scholars believe that the symptoms related to menopause are only the external appearance of CS, and "oxidative stress" is the internal basic etiology of CS. [24] With the help of multiple correlation analysis platforms and network pharmacology, in the present study, the effective components of Erzhi Pill-Erxian Decoction were explored, the target network between Erzhi Pill-Erxian Decoction and disease was constructed, and the interaction relationship therebetween was analyzed, so as to provide a reference for the study of the mechanism of "treating the same disease with different methods" for CS.The prescription of TCM for the treatment of CS is established according to the specific dialectical classification, the law is established with the syndrome, and the prescription comes from the law.Based on the analysis of the pathogenesis of CS, most compounds can regulate oxidative stress, suggesting that TCM compounds can regulate oxidative stress, that is, the same disease can be treated with different methods.Although different compatibility of medicines, the dialectical treatment is different, Erzhi Pill and Erxian Decoction can alleviate CS by regulating antioxidant targets and pathways.Multiple antioxidant receptor proteins and oxidative response inhibitor proteins are located in the downstream oxidative stress inhibition pathway.The lipid and atherosclerosis signaling pathway and the AGE-RAGE signaling pathway in diabetic complications mainly involve many complex biological processes, such as cell differentiation and apoptosis, inflammatory reaction, metabolism and so on.PI3K-Akt signaling pathway can start the gene expression of downstream phase II detoxification enzymes, antioxidant proteins, ubiquitinases and proteasomes, and improve the resistance of cells against oxidative stress. [28] previous experiment of the research group using ovarian tissue culture in vitro showed that PTEN inhibitor could activate PI3K-Akt signal pathway and activate the dormant follicles in ovarian tissue for continued development.[23] Quercetin has good superoxide quenching activity, dose-dependent can inhibit H2O2 induced cell death by up-regulating the level of Bcl-xL, and can also inhibit the production of TBARS induced by Pro oxidant drugs.The antioxidant activity of Quercetin may be mediated by Fenton reaction and negative regulation of NMDA receptor.[29] Thus, Quercetin can be used to alleviate CS.
Erzhi Pill and Erxian Decoction may be alleviating factors in CS by regulating common targets.At present, there are a large number of reports on the mechanism of Erzhi Pill and Erxian Decoction in the treatment of CS.To investigate the antioxidant activity, Chen Wuyue et al [30] measured the scavenging effect of Erzhi Pill on 2-diphenyl-2-picrylhydrazyl and hydroxyl radical in vitro.Erzhi Pill was found to have significant antioxidant activity in vitro and inhibited prednisolone induced zebrafish osteoporosis.By establishing a D-galactose induced aging cell model of Norway Rat Kidney, Liu Qinan et al [31] found that Erzhi Pill can reduce the aging degree of Norway Rat Kidney cells induced by D-galactose, and the protective effect may be related to the antioxidant stress effect of Erzhi Pill.In another study, [32] D-galactose was also used to induce aging in model rats, and the contents of mitochondrial respiratory chain complexes I and IV in rat brains were measured.Findings were made that the contents of mitochondrial respiratory chain complexes I and IV were significantly lower than those in the model control group in the Erzhi Pill treatment groups, which delayed the aging process of the body.Zhong Xunlong et al [33] showed that Erzhi Pill can improve H2O2-induced oxidative stress damage of INS-1 pancreatic islet β cells and protect their cell functions.An assumption could be made that the mechanism is also related to inhibiting the production of ROS, increasing the activity of antioxidant enzymes and protein expression.A research group of the University of Hong Kong [34] analyzed the main chemical components and pharmacological effects of Erxian Decoction, and discussed the molecular biological mechanism of the compatibility of Erxian Decoction to improve the mechanism of osteoporosis.A randomized double-blind placebo-controlled clinical trial was conducted on the efficacy of Erxian Decoction in alleviating CS, and the mechanism was studied in animal experiments.Findings were made that Erxian Decoction could regulate the secretion of estrogen, antioxidation, anti-lipid, anti-osteoporosis, reducing the metastasis of ovarian cancer cells and anti-vascular proliferation, improving the symptoms of climacteric hot flushes and night sweats, and improve the quality of life.Similarly, some scholars have conducted homologous randomized controlled trials, such as Huang et al [35] found that Erzhi Pill may improve postmenopausal-related symptoms by affecting cardiovascular risk factors as HCY.Tian et al [36] found that Erxian Decoction treatment of perimenopausal hyperlipidemia significantly relieved patients' clinical symptoms and improved their sex hormone and lipid levels.Erxian Decoction could also alleviate CS through antioxidant stress.Erxian Decoction could be a significant factor in hypothalamus, adenohypophysis, gonads and other parts, regulate the function of thalamus pituitary ovary axis, promote the increase of sexual hormones secreted by its own gonads, and improve climacteric symptoms caused by the decrease of estrogen secretion.Such results are the same as the target found in network pharmacology prediction, suggesting that Erzhi Pill and Erxian Decoction can alleviate CS by regulating the same target pathway.
Erzhi Pill can also alleviate CS by regulating specific targets, and has the effect of alleviating sub-health and antiaging.Network pharmacological studies have found that Erzhi  Pill may have used specific targets to alleviate CS.For instance, Erzhi Pill can alleviate the climacteric syndrome target APOB, with the increase of APOB indicating atherosclerosis.APOB has significant predictive value in coronary heart disease and cerebral infarction, and is closely related to the occurrence of climacteric cardiovascular disease. [37]Zhang Hezhen et al [38] found that Erzhi Pill could improve estrogen deficiency, endothelial function and lower APOB through estrogen-like effects.Additionally, Erzhi Pill can also regulate FAS ligand FASLG.FAS/FASLG can induce apoptosis and maintain the stability of the internal environment and the balance of the body.Previous studies [39] have found that the process of aging is actually a process in which the balance of the environment is broken in the body, certain cells die programmatically, and finally certain functions are damaged in the body.Compared with Erzhi Pill, Erxian Decoction has more single drugs and more specific targets for regulation.Erxian Decoction has the effects of improving blood circulation, regulating endocrine system, and resisting fatigue.The representative components of Erxian Decoction are significant factors in neuroactive ligand receptor interaction, dopaminergic synapse, cGMP-PKG signal pathway and calcium signal pathway, suggesting that Erzhi Pill and Erxian Decoction have multi-target and multi-channel characteristics.
To summarize, Erzhi Pill and Erxian Decoction are both the same and different in the treatment of CS due to their different prescriptions.Through the analysis of common targets such as AKT1, TP53, JUN, ESR1, IL1B and the biological processes and pathways where the common targets are located, the "same disease" of "treating the same disease with different methods" for CS is clarified.AKT1 is widely present in ovarian cells and plays an emphatic role in regulating follicular growth and development, inhibiting apoptosis and maintaining normal follicular cycles. [40]TP53 is also associated with apoptosis or death of ovarian granulosa cells. [41]JUN induces apoptosis in ovarian granulosa cells and inhibits estrogen production, leading to follicular dysplasia. [42]ESR1 is widely present in the HPG axis and directly binds to estrogen. [43]It has been demonstrated that reducing ovarian inflammation through anti-inflammation and decreasing IL1B expression can improve ovarian decline. [44]t is hypothesized that the key targets of the 2 prescriptions for the treatment of CS mainly involve cell proliferation, apoptosis, hormonal effects, anti-inflammatory processes.APOB, CYP19A1, FASLG, ABCC2, ADRA2A, ADRA2B, and other specific targets and the biological process and pathway analysis of the specific targets preliminarily explain the dialectical connotation of "different treatment" for CS.It has been shown that higher blood low-density lipoprotein (LDL) containing apolipoprotein B (apoB) in middle age is associated with an increased risk of Sporadic Alzheimer's Disease (SAD) in later life, which has led to the nomination of LDL as an important discriminating factor in the etiology of dementia. [45]Other studies have shown that elevated CYP19A1 expression may be a marker of improved granulosa cell function, granulosa cell proliferation and prevention of ovarian hypoplasia. [46]It has also been found that both men and women over 50 years of age have high expression of FASLG compared to younger people (20-30 years of age), which may induce a hyperinflammatory cascade of activation of specific immune cells. [47]It has also been found that the ABCC2 gene may be involved in the regulation of bile acid transport and cholesterol metabolism, thus affecting the homeostasis of lipid metabolism. [48]The gene for adrenergic receptor ADRA2A downregulated with age has a rich set of biological functions, most notably involvement in pathways associated with neurons, dendrites, synapses, and neuronal receptors and ion channels. [49]Whereas brain aging may be naturally inhibited by adenosine receptor A2B (ADORA2B), a protein on the red blood cell membrane known to contribute to the release of oxygen from blood cells, thus making it available to the body. [50]The aforementioned predicted targets are consistent with the pharmacological effects reported in previous literature, indicating the accuracy of the predicted targets.Further, there is a scarcity of reports on the remaining targets in the aforementioned discussion, which can provide a direction for the further study of the molecular mechanism of potential targets in alleviating CS in the future.
Through the network pharmacological analysis of "treating the same disease with different methods" of classical prescriptions for CS, as a TCM compound, there are the same and different targets in the treatment of CS.Such findings show the multi-component, multi-target and integrated regulation characteristics of the prescriptions.There are still many limitations in this study.At this stage, the understanding of the pathogenesis of CS is not comprehensive, and the preventive measures are not comprehensive.At the same time, the database and software as well as the method itself have certain limitations.There will be certain differences between the calculation results and clinical efficacy, which needs further research.In view of the above problems, the TCM compound for the treatment of clinical CS is different according to different types of CS.An explanation is also needed for the correlation mechanism between prescriptions and syndromes in combination with the pharmacodynamic substances of the compound.The mechanism of classical ancient prescriptions needs to be further studied in the treatment of CS.

Figure 1 .
Figure 1.The study flow chart.

Figure 4 .
Figure 4. Common or unique chemical constituents of Erzhi Pill and Erxian Decoction-CS target interaction network.CS = climacteric syndrome.

Figure 5 .
Figure 5. PPI network of intersection target of Erzhi Pill-Erxian Decoction and CS.(A) The network action diagram of common targets.(B) The specific network action diagram of anti-inhibition targets of Erzhi Pill.(C) The specific network action diagram of anti-inhibition targets of Erxian Decoction.CS = climacteric syndrome, PPI = protein-protein interaction network.

Figure 6 .
Figure 6.GO enrichment annotation and KEGG pathway enrichment analyses.(A) Enriched KEGG pathways of potential targets from main active ingredients of.(B) Enriched KEGG pathways of potential targets from main active ingredients of Erzhi Pill-Erxian Decoction.(C) The lipid and 4 atherosclerosis, the AGE-RAGE signaling pathway in diabetic complications and PI3K-Akt signaling pathway.GO = gene ontology, KEGG = Kyoto encyclopedia of genes and genomes.

Table 8
Core targets and molecular docking results of main active components.

Table 1
Information of ingredients number and targets number from Erzhi Pill and Erxian Decoction.
Figure 2. Basic information of composition and target from Erzhi Pill and Erxian Decoction.

Table 2
Basic information of active compounds form Erzhi Pill and Erxian Decoction.
kinds in Privet; 204 corresponding targets were found.There were 69 kinds of effective chemical components found in Erxian Decoction, including 15 kinds in Morinda officinalis, 2 kinds in Angelica sinensis, 23 kinds in Golden cypress, 6 kinds in Star grass, 22 kinds in Longspur epimedium and 12 kinds in Rhizoma anemarrhenae; 233 corresponding targets were found.The basic information of Erzhi Pill and Erxian Decoction are shown in Tables the TCMSP database searched by OB and DL, Erzhi Pill had 16 active ingredients, including 9 kinds in Eclipta and 9

Table 3
Analysis of topological properties of dominant targets in ErzhiPill (top 10 mediators).

Table 4
Analysis of topological properties of dominant targets in ErxianDecoction (top 10 mediators).

Table 5
Analysis of topological properties of Erzhi Pill and ErxianDecoction (top 10 mediators).

Table 6
Modular analysis of common targets of Erzhi Pill and ErxianDecoction.

Table 7
Pathway analysis of common targets of Erzhi Pill-Erxian Decoction.